Process for producing acrylamide using a microbial catalyst having been washed with aqueous acrylic acid solution

ABSTRACT

Provided is a process for producing acrylamide with good storage stability and improved acrylamide polymer physical properties using a microbial catalyst. A microbial catalyst having catalytic activity to convert from acrylonitrile to acrylamide is washed with an aqueous acrylic acid solution, and then the washed microbial catalyst is used for the conversion reaction, so that the production of the above acrylamide is achieved.

TECHNICAL FIELD

The present invention relates to a process for producing acrylamide fromacrylonitrile by the action of a microorganism-derived enzyme,nitrilehydratase. Acrylamide is used in a variety of fields as anindustrially important substance. For example, acrylamide polymers arewidely used in applications such as a coagulant for wastewatertreatment, a paper strong agent, an oil collecting agent and the like.

BACKGROUND ART

Acrylamide has been conventionally produced industrially by hydratingacrylonitrile corresponding thereto using copper in the reduced state asa catalyst. Recently, a method using a microbial catalyst instead of acopper catalyst has been developed, and a part of this method is inactual use. A biocatalytic method using a microbial catalyst or the likehas promise as an industrial production method, because the method hasmoderate reaction conditions and yields almost no by-product, so that anextremely simple process can be designed for this method. Thus, manymicroorganisms having an enzyme (enzyme name: nitrilehydratase) capableof catalyzing (converting) acrylonitrile into acrylamide by hydrationhave been found so far.

Examples of these microorganisms include microbial strains belonging tothe genera Bacillus, Bacteridium, Micrococcus, Brevibacterium [see JPPatent Publication (Kokoku) No. 62-21519 B (1987) for the abovemicroorganisms], Corynebacterium and Nocardia [see JP Patent Publication(Kokoku) No. 56-17918 B (1981) for the above microorganisms],Pseudomonas [see JP Patent Publication (Kokoku) No. 59-37951 B (1984)],Rhodococcus and Microbacterium [see JP Patent Publication (Kokoku) No.4-4873 B (1992) for the above microorganisms], Rhodococcus rhodochrous[see JP Patent Publication (Kokoku) No. 6-55148 B (1994)], andRhodococcus [see JP Patent Publication (Kokoku) No. 7-40948 B (1995)].

Examples of a process for producing acrylamide using the abovemicroorganism as a microbial catalyst include those of JP PatentPublication (Kokai) Nos. 11-123098 A (1999) and 7-265091 A (1995), andJP Patent Publication (Kokoku) No. 56-38118 B (1981). An example of thereaction method is that of JP Patent Publication (Kokai) No. 11-89575 A(1999).

Further, a variety of studies have been conducted for improving enzymeactivity or suppressing a decrease (deactivation of) in enzyme activityduring reaction. Examples of such a study include a process whichinvolves performing reaction at a low temperature, 15° C. below freezingpoint [see JP Patent Publication (Kokoku) No. 56-38118 B (1981)], aprocess which involves sequentially supplying a substrate at a lowconcentration from multiple supply openings [see JP Patent Publication(Kokoku) No. 57-1234 B (1982)], a process which involves treatingmicroorganisms or the treated product thereof with an organic solvent[see JP Patent Publication (Kokai) No. 5-308980 A (1993)] a processwhich involves performing reaction under the presence of higherunsaturated fatty acid [see JP Patent Publication (Kokai) No. 7-265090 A(1995)], and a process which involves subjecting microbial cells tocross-linking treatment with glutaraldehyde or the like [see JP PatentPublication (Kokai) Nos. 7-265091 A (1995) and 8-154691 A (1996)].

In the meantime, for washing a microbial catalyst, the generally knownmethods involve washing using a physiological saline, a buffer such asan aqueous solution of phosphate or Tris hydrochroride to suppress adecrease in enzyme activity. However, there is no report on the washingof a microbial catalyst wherein the effects of wash components on thephysical properties of acrylamide polymers and the storage stability ofmonomers have been considered.

As described above, a process for producing acrylamide using a microbialcatalyst has promise as an industrial production process, because theprocess employs moderate reaction conditions and yields almost noby-product, so that no purification is required and an extremely simpleprocess may be designed.

Although the above production processes yield no by-product upon enzymereaction, they have a drawback such that when a microbial catalyst to beused is washed, contamination of impurities derived from the washaffects the physical properties of acrylamide polymers and the storagestability of acrylamide monomers. To address the problem, purificationsuch as crystallization, ion exchange, or distillation can be performed.With these purification processes, however, an outstandingcharacteristic of a production process using a microbial catalyst, thatis, to yield almost no by-product upon reaction, cannot be utilized.Moreover, the use of these processes is also unfavorable in terms ofenergy and environmental problems.

SUMMARY OF THE INVENTION

As a result of intensive studies to address the above problems, we havecompleted the present invention by finding that acrylamide with improvedphysical properties of acrylamide polymers and improved storagestability of acrylamide monomers can be produced by using a microbialcatalyst washed with an aqueous acrylic acid solution in a process forproducing acrylamide from acrylonitrile using a microbial catalysthaving a microorganism-derived enzyme, nitrilehydratase.

In other words, the present invention is a process for producingacrylamide using a microbial catalyst that converts acrylonitrile toacrylamide, which uses the microbial catalyst having been washed with anaqueous acrylic acid solution.

The microbial catalyst that can be used in the present invention may beany catalyst as long as it is prepared from microorganisms havingcatalytic activity (nitrilehydratase activity) to convert acrylonitrileto acrylamide. Preferred examples of such microbial species includethose belonging to the genus Bacillus, genus Bacteridium, genusMicrococcus, genus Brevibacterium, genus Corynebacterium, genusNocardia, genus Pseudomonas, genus Microbacterium, genus Rhodococcus,genus Achromobacter, and genus Pseudonocardia. One of or a combinationof these microorganisms can be used.

Further, a transformant that may be used herein is prepared by obtaininga nitrilehydratase gene derived from the above microorganism, and thenintroducing the gene directly, or the artificially improved gene, into afreely chosen host.

Preferred examples of the above transformant include Escherichia coliMT10770 (FERM P-14756) (JP Patent Publication (Kokai) No. 8-266277 A(1996)) that has been transformed with nitrilehydratase of the genusAchromobacter, Escherichia coli MT10822 ((FERM BP-5785) (JP PatentPublication (Kokai) No. 9-275978 A (1997)) that has been transformedwith nitrilehydratase of the genus Pseudonocardia, or microorganismstransformed with nitrile-hydratase (JP Patent Publication (Kokai) No.4-211379 A (1992) of the genus Rhodococcus rhodochrous.

The above microorganisms can be cultured by any method that isappropriate for a given microbial species.

In the present invention, the microbial catalyst that is prepared frommicroorganisms refers to a culture solution obtained by culturingmicroorganisms, cells obtained by a harvesting process or the like,cells disrupted by ultrasonication or the like, or those prepared aftercell disruption including a crude enzyme, a partially-purified enzyme ora purified enzyme. If necessary, these microbial catalysts may beimmobilized on carriers such as polyacrylamide gel, alginate,carrageenan or ion exchange resin. A mode to use the microbial catalystmay be appropriately selected depending on enzyme stability, productionscale and the like.

The term “washing” in the present invention refers to the washing ofmicrobial cells that have been cultured and/or microbial catalysts to beused in a reaction. Thus, both microbial cells that have been culturedand microbial catalysts to be used in a reaction may be washed withacrylic acid, or only the microbial catalysts to be used in a reactionmay be washed with acrylic acid. For example, a microbial catalyst to beused in a reaction may be washed once with water, a buffer or the like,and then washed with acrylic acid before the reaction. Microbialcatalysts may be washed with acrylic acid immediately before thereaction.

Further, any washing method can be employed. Examples of such a methodthat can be illustrated herein include a method which involves repeatedwashing and centrifugation, and a washing method using a hollow fibermembrane. Further, immobilized microbial catalysts can be washed byrepeating agitation and precipitation of the immobilized catalysts in awash and the removal of supernatant.

Any washing method and any number of washing can be appropriately set inconsideration of washing efficiency, enzyme stability and the like.

The concentration of acrylic acid to be used for washing is preferablybetween 0.01% by mass and 10% by mass in an aqueous acrylic acidsolution. More preferably, the concentration is between 0.05% by massand 1% by mass, and most preferably is 0.1% by mass.

When the concentration of acrylic acid is 0.01% by mass or less, theduration of washing and the number of washing increase, so as to makethe procedures complex. Furthermore, such increased number of washingcause cell disruption during washing, collapsed immobilized cells andthe like. 10% by mass or more of the concentration is unfavorablebecause it causes a decrease in enzyme activity, and is also unfavorableeconomically.

The pH of an aqueous acrylic acid solution is adjusted using sodiumhydroxide, ammonia or the like. Preferably the pH of the solution usedherein is adjusted to be between 5 and 11, more preferably between 6 and10, and most preferably to be 7.

Microbial catalysts prepared as described above can be used as microbialcatalysts in a state of suspension or dispersal in an aqueous acrylicacid solution, or in a state of being subjected to solid-liquidseparation.

BEST MODE OF CARRYING OUT THE INVENTION

The present invention will be described more specifically by thefollowing examples. These examples are not intended to limit the scopeof the present invention.

EXAMPLE 1 (1) Process for Preparing Cultured and Washed Microbial Cells

Rhodococcus rhodochrous J-1 (FERM BP-1478) having nitrilehydrataseactivity (JP Patent Publication (Kokoku) No. 6-55148 B (1994)) wascultured in a medium (pH 7.0) containing 2% by mass of glucose, 1% bymass of urea, 0.5% by mass of peptone, 0.3% by mass of yeast extract,and 0.05% by mass of cobalt chloride in a 30 L jar fermenter (TakasugiSeisakusho) at 30° C. for 60 hours aerobically.

20 liters of the solution cultured as described above were filtered bycirculation through a cross flow type hollow fiber membrane module. Thecultured cells were washed by sequentially supplying 0.7% by mass ofphosphate buffer (pH 7.0) in a volume corresponding to the volume of thefiltrate to the culture solution, thereby obtaining washed microbialcells.

(2) Preparation of Microorganism-Immobilized Carriers

To 500 g of the washed cell suspension (20% by mass when converted intodry cell weight) obtained in (1), 500 g of a monomer mixture solutioncontaining acrylamide, methylene bisacrylamide and2-dimethylaminopropylmethacrylamide with a concentration of 20%, 2% and2% by mass, respectively, was added, so as to perform suspension well.5% by mass (2 g) of ammonium persulfate and 50% by mass (2 g) ofN,N,N,N-tetramethylethylenediamine were added to the suspension forpolymerization and gelatinization. The product was cut into anapproximately 1-mm cube, thereby obtaining microorganism-immobilizedcarriers.

The microorganism-immobilized carrier obtained by the above method wassubjected to 20 cycles of a procedure, each cycle consisting of thefollowing steps: (1) suspension and agitation in a 0.1% by mass aqueoussodium acrylate solution (pH 7.0), (2) still standing and precipitation,and (3) disposal of supernatant.

(3) Amidation Reaction

3200 g of a 0.2 g/L aqueous sodium acrylate solution was put in aseparable flask with an internal volume of 5 liters. To the aqueousacrylic acid solution, 3 g of the immobilized microorganisms prepared in(2) was added. The solution was agitated while maintaining pH 7.0 and atemperature of 20° C.

To this solution, acrylonitrile was sequentially fed for keeping theconcentration of acrylonitrile at 2% by mass, and then an accumulationreaction was performed until the acrylamide concentration became 50% bymass.

After the end of the reaction, the solution was filtered through amembrane filter with an aperture of 0.45 μm, so as to remove thecatalyst.

(4) Method for Evaluating the Physical Properties of Polymer

The 20% by mass of acrylamide obtained in Example 1(3) was dissolved in80% by mass of water. After the pH was adjusted to 8.0, the solution wastransferred into a Dewar flask, and then the air within the system wasreplaced by nitrogen. Then, 0.0004% of ammonium persulfate, 0.0004% ofiron sulfate, and 0.01% of 4,4′-azobis-(4-cyanovaleric acid) were addedto perform polymerization. The thus obtained water-containing gelatinouspolymers were shredded into particles with a diameter of several mmusing a meat mincer. The particles were then dried at 80° C. for 10hours, and then disrupted to have a particle size of 2 mm or less usinga Wiley grinder, thereby obtaining polymer powder.

The thus obtained polymer powder was prepared to have a concentration of0.2% with 500 g of water. The solution was agitated at room temperaturefor 4 hours and then dissolved. The Brookfield viscosity (type Bviscometer, the number of rotation of a rotor: 30 rpm, and Rotor No. 1)was then measured. Then, the solution was filtered through a 80-meshwoven metal wire, and then the mass of insoluble matters that hadremained on the wire after washing with water was measured.

(5) Method for Evaluating Monomer Storage Stability

50 g of the 50% acrylamide monomer aqueous solution obtained in Example1 (3) and iron test pieces were put in a 100 ml polyethylene-madebottle, and then the bottle was closed with a cap to avoid evaporation.This bottle was stored in a high temperature box at 50° C., and then thestability was determined based on the presence or absence of polymerizedproducts.

The results of Example 1 (4) and (5) are shown in Table 1 and Table 2.

COMPARATIVE EXAMPLE 1

Comparative example 1 was performed similarly to Example 1 except forusing a 0.7% phosphate buffer (pH 7.0) instead of 0.1% by mass ofaqueous sodium acrylate solution (pH 7.0) in the step of preparingmicroorganism-immobilized carriers in Example 1 (2). The results areshown in Table 1 and Table 2.

TABLE 1 Physical properties of polymer Time for Insoluble polymerizationViscosity matter [min.] [mPa · s] [g] Example 1 17 44 0 Comparative 74150 290 example 1

TABLE 2 Storage stability Days of storage [day] Example 1 >10Comparative <1 example 1

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

As described in detail above, the use of a microbial catalyst that hasbeen washed with acrylic acid when acrylamide is produced using amicrobial catalyst makes it possible to obtain acrylamide with highstorage stability and good quality.

1. A process for producing acrylamide comprising washing a microbialcatalyst comprising a microorganism having nitrile hydratase activitywith a solution consisting of acrylic acid and water, and thencontacting the washed microbial catalyst with acrylonitrile.
 2. Theprocess for producing acrylamide according to claim 1, wherein themicrobial catalyst is prepared from at least one microorganism selectedfrom the group consisting of the genus Bacillus, genus Bacteridium,genus Micrococcus, genus Brevibacierium, genus Corynebacterium, genusNocardia, genus Pseudomonas, genus Microbacterium, genus Rhodococcus,genus Achromobacter, and genus Pseudonocardia.
 3. The process forproducing acrylamide according to claim 1, wherein the concentration ofacrylic acid in the aqueous acrylic acid solution is between 0.01% bymass and 10% by mass.
 4. The process for producing acrylamide accordingto claim 1, wherein the pH of the aqueous acrylic acid solution isbetween 5 and
 11. 5. The process for producing acrylamide according toclaim 2, wherein the concentration of acrylic acid in the aqueousacrylic acid solution is between 0.01% by mass and 10% by mass.
 6. Theprocess for producing acrylamide according to claim 2, wherein the pH ofthe aqueous acrylic acid solution is between 5 and
 11. 7. The processfor producing acrylamide according to claim 3, wherein the pH of theaqueous acrylic acid solution is between 5 and
 11. 8. The processaccording to claim 1, wherein the microbial catalyst is an unimmoblizedmicroorganism comprising a nitrile hydratase gene.